Anthrax is an old disease with re-born relevance to both scientists and public. To fight this old foe, the causative organism, Bacillus anthracis, must be understood clearly and fully at the mechanistic molecular level of its pathogenesis. Many Gram-positive pathogenic bacteria possess cell wall anchored proteins (CWAPs) that are critical for virulence and are excellent vaccine candidates. By analogy, the CWAPs of B. anthracis are likely to show equal relevance and utility. Analysis of the B. anthracis genome revealed nine previously uncharacterized CWAPs. Preliminary studies indicated a recombinant fragment of one of these recognized specific macrophage targets. Macrophages play a central role in the establishment of anthrax. Two additional CWAPs were found to bind collagen, which is a major component of the skin where cutaneous anthrax develops. Together these findings support the hypothesis that CWAPs of B. anthracis are significant in its pathogenesis. The two specific aims of this proposal are to determine the roles of CWAPs in the interaction of B. anthracis with 1) macrophages and 2) major skin components i.e., collagen and fibroblasts. To achieve these aims, deletion mutants will be generated for each of the nine CWAPs. The mutants will be evaluated for their ability to associate with, be engulfed by and survive within macrophages, as well as their ability to adhere and invade human dermal fibroblasts. The respective mutants will also be used to determine the relevance of each of the two collagen-binding CWAPs in the adherence of B. anthracis to collagen. To confirm the function of the CWAPs, the deleted genes will be complemented then expressed in a heterologous host. Their molecular targets in the host cells will subsequently be identified. In the future, the effect of these proteins in B. anthracis virulence will be evaluated in an animal model in collaboration with Dr. Theresa Koehler at University of Texas Medical School, and Dr. Rick Lyons at University of New Mexico. The long-term objectives are to elucidate the biological functions of these proteins, their molecular interactions with the host and their potential as vaccine and drug targets. The information will likely improve the understanding of the infection mechanisms of B. anthracis, and may provide novel effective ways to combat the scourge of anthrax.